Electrospray ionization mass spectrometry (ESI-MS) combined with multiple tandem mass spectrometry (MS(n)) has been applied for simultaneously determining lipo-alkaloids from the aconite tubers. ESI-MS can give a very good mass separation for lipo-alkaloid mixture and MS(n) can provide structural information. Accordingly, structures were assigned to 12 known lipo-alkaloids including lipoaconitines, lipomesaconitines, lipodeoxyaconitines, and lipohypaconitines on the base of the information obtained using MS(n). Also, four new lipo-alkaIoids were found and their structures were interpreted using the MS(n) data.